Many types of sample analysis rely upon the fluorescence properties of a stain. Fluorescence occurs when a molecule excited by light of one wavelength returns to the unexcited (ground) state by emitting light of a longer wavelength. The exciting and emitted light, being of different wavelengths, can be separated from one another using optical filters. Fluorescence has been used to visualize certain molecules (and hence structures) by light microscopy for many years, and is also used in other analytical techniques, such as flow cytometry.
The type of fluorescent probe used in fluorescent analysis can be divided into two broad categories, those used to label covalently other probes (often antibodies) and those whose distribution or fluorescence reflects their environment and hence particular properties of a cell. Among the latter, fluorescent compounds that bind specifically to nucleic acids or to cytoskeleton structures or elements are particularly important.
A variety of fluorescent probes are known. For example, propidium and ethidium stains are available. These compounds, however, bind to both deoxyribonucleic acid (DNA) and double stranded ribonucleic acid (RNA). Thus, RNA has to be removed if DNA is to be measured.
4', 6-diamidino-2-phenylindole (DAPI) is also used as a DNA stain for numerous applications in cytology, nucleic acid biophysical analysis, and flow cytometry. DAPI binds preferentially to the DNA minor groove at AT-rich DNA sequences but also intercalates at GC and mixed GC/AT sequences, and significantly binds RNA. W. D. Wilson, et al., "The Effects of Ligand Structure on Binding Mode of Unfused Aromatic Cations with DNA" in Molecular Basis of Specificity in Nucleic Acid-Drug Interactions, Klumar Academic Publishers, Amsterdam (1990), pps. 331-353; W. D. Wilson, et al., Biochemistry 32, 4098-4104 (1993). DAPI also binds other cellular components, such as tubulin, resulting in RNA and tubulin staining.
A number of aryldiamidines have been synthesized in the past with value as anti-protozoal agents. B. P. Das and D. W. Boykin, J. Med. Chem. 20, 531-536 (1977). Like DAPI, 2,5-bis(4-amidinophenyl) furan (also referred to as 2,5-bis(4-guanylphenyl) furan) binds preferentially to the DNA minor groove at AT-rich DNA sequences but also interacts with GC and mixed GC/AT sequences. W. D. Wilson, et al. (1990), supra; W. D. Wilson, et al. (1993), supra.
Another stain used in fluorescent analysis applications is the bis-benzimide Hoechst 33258. Loontiens et al have suggested that Hoechst 33258, a closely related molecule, binds to GC-rich sequences via a self-association complex in the major groove of DNA. F. G. Loontiens, et al., Biochemistry 29, 9029-9039 (1990). This agent also has a significant association with RNA. Wilson, et al. (1993), supra. Thus, as with DAPI and 2,5-bis(4-amidinophenyl)furan, significant staining of non-DNA elements can result with Hoechst 33258.